Methods and means for characterizing antibiotic resistance in microorganisms

ABSTRACT

The present invention relates to a method for characterizing the antibiotic resistance of a microorganism, said method comprising the steps of (a) providing a reference mass spectrum of an antimicrobial compound, its enzymatic modification product, its molecular target, or of a substrate compound of a its modifying enzyme; (b) exposing a microorganism, a cell lysate thereof, or a growth medium supernatant thereof, to said antimicrobial compound or said substrate compound in aqueous liquid to thereby provide an exposed sample; (c) acquiring a mass spectrum of the exposed sample; (d) comparing the mass spectrum acquired in step c) with the reference mass spectrum of step (a), and (e) determining from said comparison whether modification of said antimicrobial compound, its modification product or its molecular target or of said substrate has occurred following said exposure, and establishing that said microorganism is potentially resistant to said antimicrobial compound when said modification is observed.

PRIORITY INFORMATION

This patent application claims priority from PCT patent applicationPCT/NL2010/050523 filed Aug. 19, 2010, which is hereby incorporated byreference.

FIELD OF INVENTION

The present invention is in the field of bacterial diagnostics andrelates in particular to a method for characterizing the antibioticresistance of a microorganism, to a kit of parts for performing themethod of the invention and to a system for characterizing theantibiotic resistance of a microorganism comprising a mass spectrometricdevice and sample preparation materials for performing the method of theinvention.

BACKGROUND OF THE INVENTION

Antibiotic resistance is the ability of a microorganism to withstand theeffects of an antibiotic. This resistance develops through geneticmutation and plasmid exchange between microorganisms. Already,antibiotic resistance is having a major impact on medicine that willonly increase in the coming years.

One group of the opportunistic microorganisms that gain renewed interestfor exhibiting antibiotic resistance are the Enterobacteriaceae. Thesebacterial species (including for example Klebsiella spp and Escherichiacoli) comprise opportunistic pathogens that have i. a. been associatedwith urinary tract infections, septicaemia, respiratory tract infectionsand diarrhoea. Resistance of these species to third generationcephalosporins such as oxyimino beta-lactams has been known for 30 yearsbut an exponential increase in resistance has since been recorded.Strains gain their resistance by producing so-called extended-spectrumbeta-lactamases (ESBLs), which are Molecular Class A beta-lactamases,capable of inactivating third-generation cephalosporins (ceftazidime,cefotaxime, and cefpodoxime) as well as monobactams (aztreonam). ESBLsare derivatives of common beta-lactamases (e.g. TEM and SHVbeta-lactamases) that have undergone one or more amino acidsubstitutions near the active site of the enzyme, thus increasing theiraffinity for and hydrolytic activity against third generationcephalosporins and monobactams. Extensive use of newer generationcephalosporins drives the evolution of an increasing range of new ESBLs.ESBLs are encoded by transferable conjugative plasmids that areresponsible for the dissemination of resistance to other members of gramnegative bacteria.

ESBLs are distinguished into more than 450 types based on their physicalproperties and are variably inhibited by clavulanate, sulbactam andtazobactam, a property which has been used to detect them in thelaboratory. Currently, only phenotypic ESBL detection tests are used inthe clinical microbiology laboratory. Molecular (genotypic) tests areunder development. A problem with molecular tests is, however, the lackof a 100% correlation between the genotype and the phenotype. Hence, thepredictive value of molecular testing for any bacterial phenotype,including ESBL producing bacteria, is limited.

In general, the current phenotypic laboratory tests are sensitive andspecific as compared to ESBL genotypic confirmatory tests. Allphenotypic ESBL detection tests rely on the same principle: the testsassess variation in the inhibition of bacterial growth in the presenceof beta-lactam antibiotics or combinations of beta-lactam antibioticsand beta-lactamase inhibitors. Various manual tests and automatedplatforms are commercially available for performing these phenotypictests. The manual tests use disks or strips impregnated with beta-lactamantibiotics or combinations of beta-lactam antibiotics andbeta-lactamase inhibitors. The impregnated material is placed on solidmedia that is pre-inoculated with a bacterial suspension of knowndensity. Following overnight incubation, growth inhibition is determinedvisually and can be quantified on the basis of the diameter ofinhibition zones. The automated systems are also based on measurement ofbacterial growth in the presence of panels of beta-lactam antibiotics orcombinations of beta-lactam antibiotics and beta-lactamase inhibitors atdifferent concentrations. Results of such systems are obtained after 4h-18 h.

There is presently a need for means and methods that are capable ofdiagnosing ESBL producing bacteria more rapidly. There is also a needfor an ESBL detection test that can be used in the clinical microbiologylaboratory to characterize the ESBL enzymes in terms of enzyme kineticsin order to track evolutionary trends and evaluate and predict theeffective dosage in antibiotic therapy. Preferably, such means findwider applicability in characterizing antibiotic resistance inmicroorganisms in general.

SUMMARY OF THE INVENTION

The present invention now provides means and methods for rapid diagnosisof antibiotic modifying enzyme-producing microorganisms, in particular(in preferred embodiments) microorganisms that produce ESBL. The presentinvention further provides means and methods for characterizing theantibiotic modifying enzymes themselves. Such characterization may leadto an earlier detection of novel types of resistance.

In a first aspect, the present invention provides a method forcharacterizing the antibiotic resistance of a microorganism, said methodcomprising the steps of:

-   -   a) providing a reference mass spectrum of an antimicrobial        compound, its enzymatic modification product, its molecular        target, or of a substrate compound of a its modifying enzyme;    -   b) exposing a microorganism, a cell lysate thereof, or a growth        medium supernatant thereof, to said antimicrobial compound or        said substrate compound in aqueous liquid to thereby provide an        exposed sample;    -   c) acquiring a mass spectrum of the exposed sample;    -   d) comparing the mass spectrum acquired in step c) with the        reference mass spectrum of step a), and    -   e) determining from said comparison whether modification of said        antimicrobial compound, its modification product or of said        substrate or whether overproduction of its molecular target has        occurred following said exposure, and establishing that said        microorganism is potentially resistant to said antimicrobial        compound when said modification is observed.

In a preferred embodiment of said method, the modification comprisesenzymatic inactivation or enzymatic degradation of said antimicrobialcompound, and/or methylation or overproduction of its molecular target.More preferably, the enzymatic degradation is due to degradation by abeta-lactamase. In such instance, the antimicrobial compound may be abeta lactam antibiotic or any other beta-lactamase substrate. Hence, inanother preferred embodiment of said method, the antimicrobial compoundmay be replaced by a substrate compound of an enzyme that modifiesantimicrobial compounds. In other preferred embodiments theantimicrobial compound is a beta-lactam antibiotic, preferably selectedfrom the group consisting of penicillins, cephalosporins, cephamycins,and carbapenems, more preferable selected from the group consisting ofceftazidime, cefotaxime, ceftriaxone, cefpodoxime, and aztreonam.

It is clear that, depending on the mechanism of antibiotic resistance,also the molecular target of the antimicrobial compound (such as forinstance folate) may be overproduced, which results in resistance tofolate antagonists. Target overproduction may be detected by usinginternal standards and observing that the ratio of target/internalstandard is increased. Suitable internal standards may be nucleic acidssuch as DNA.

Further, again depending on the mechanism of antibiotic resistance, alsothe methylation of the molecular target of the antimicrobial compound(such as for instance nucleic acid) may be detected.

In yet another preferred embodiment of a method of the invention, themethod is performed by exposing said microorganism to multipleantimicrobial compounds simultaneously, thereby characterizing theantibiotic resistance of said microorganism for multiple antibioticcompounds.

In yet another preferred embodiment of a method of the invention, theenzymatic inactivation or enzymatic degradation of said antimicrobialcompound is brought about by a beta-lactamase. In particularly preferredembodiments, the beta-lactamase enzyme may be selected from the groupconsisting of cephalosporinases (including extended spectrumcephalosporinases), penicillinases, carbenicillinases, cloxacillinasesand carbapenemases.

In still a further preferred embodiment of a method of the invention,the beta lactamase enzyme is an extended-spectrum beta lactamase (ESBL).

In still a further preferred embodiment of said method, themicroorganism is a suspected ESBL-producing microorganism, preferably aGram-negative bacterium, more preferably a Gram-negative bacteriumselected from Klebsiella pneumoniae, Escherichia coli, Klebsiellaoxytoca and Proteus mirabilis.

The samples used in aspects of the invention comprise microorgansisms orlysis products thereof. The samples of microorganisms may be samples ofcultures of microorganisms. Such cultures need not be pure cultures.Alternatively, also fractions of culture media or direct clinicalmaterials can be a source of the sample.

In yet another preferred embodiment of said method, the method is partof a method for characterization of an antibiotic modifying enzyme of amicroorganism, preferably said antibiotic modifying enzyme is anextended-spectrum beta lactamase (ESBL) enzyme. In a particularlypreferred embodiment, the method of the invention is part of a methodfor characterization of an extended-spectrum beta lactamase (ESBL)enzyme.

Preferably, the method for characterization of said enzyme according tothe invention comprises the determination of the rate of modification,preferably degradation, of said antimicrobial compound or said substratecompound with or without the presence of specific enzymatic inhibitorsand/or the rate of production of the enzymatic modification product ofsaid compound or substrate or the rate of overproduction of themolecular target of said compound to thereby determine theMichaelis-Menten (Km) constant and maximum reaction rate (Vmax) for saidenzyme.

In yet another preferred embodiment of said method, the mass spectra areacquired using MALDI triple-quadrupole Mass Spectrometry.

In yet another preferred embodiment of said method, the exposed sampleis an exposed crude cell lysate of said microorganism.

In yet another preferred embodiment of said method, the method furthercomprises the step of quantifying the microorganism. Preferably, themicroorganism is quantified by quantifying in said samples one or morestructural biomolecules or metabolites derived from said microorganism.In preferred embodiments, the structural biomolecules or metabolites areselected from the group consisting of nucleic acids, preferably(genomic) DNA. DNA is present as a single molecule inside the cell andcan be quantified using for instance PCR- and/or DNA probing mediatedtechnologies.

In another aspect, the present invention provides a kit-of-parts forcharacterizing the beta-lactam antibiotic resistance of a microorganismcomprising:

-   -   a) a lysis buffer for lysing a microorganism;    -   b) at least one antimicrobial compound or a substrate of an        antimicrobial compound-modifying enzyme, and    -   c) a MALDI matrix material,        preferably said kit of parts further comprising:    -   d) a carrier carrying said at least one antimicrobial compound        or substrate, wherein said carrier is optionally in the form of        a disposable mass spectrometric sample support.

In yet another aspect, the present invention provides a system adaptedfor characterizing the beta-lactam antibiotic resistance of amicroorganism by a method of the invention as described above, saidsystem comprising one or more of the following:

-   -   at least one antimicrobial compound or a substrate of an        antimicrobial compound-modifying enzyme;    -   a container for exposing a microorganism, a cell lysate thereof,        or a growth medium supernatant thereof, to said at least one        antimicrobial compound in aqueous liquid, preferably wherein        said at least one substrate compound is provided in said        container;    -   a lysis buffer for lysing said microorganism;    -   a MALDI matrix material;    -   a mass spectrometry device;    -   a reference mass spectrum of an antimicrobial compound, its        enzymatic modification product, its molecular target, or of a        substrate compound of a its modifying enzyme, and    -   a mass spectrometric sample support,        optionally further comprising    -   an automated pipetor for liquid handling;    -   a computer program comprising computer program code means for        performing all the steps of the method of the invention as        described above when said program is run on a computer including        for instance algorythms for results interpretation, interface        software and/or expert system software.

The invention in another aspect provides a computer program comprisingcomputer program code means for performing all the steps of the methodof the invention as described above when said program is run on acomputer.

In another aspect the invention provides a computer program productcomprising computer program code means stored on a computer readablemedium for performing the method of the invention as described when saidprogram product is run on a computer.

These and other objects, features and advantages of the presentinvention will become more apparent in light of the following detaileddescription of preferred embodiments thereof, as illustrated in theaccompanying drawings.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms “antibiotic” and “antimicrobial compound” are usedinterchangeably herein and are used herein to describe a compound orcomposition which decreases the viability of a microorganism, or whichinhibits the growth or reproduction of a microorganism “Inhibits thegrowth or reproduction” means increasing the generation cycle time by atleast 2-fold, preferably at least 10-fold, more preferably at least100-fold, and most preferably indefinitely, as in total cell death. Asused in this disclosure, an antibiotic is further intended to include anantibacterial, bacteriostatic, or bactericidal agent. Non-limitingexamples of antibiotics useful in aspect of the invention includepenicillins, cephalosporins, aminoglycosides, sulfonamides, macrolides,tetracyclins, lincosamides, quinolones, chloramphenicol, glycopeptides,metronidazole, rifampin, isoniazid, spectinomycin, folate inhibitors,sulfamethoxazole, and others.

The term “beta-lactam antibiotic” is used to designate compounds withantibiotic properties containing a beta-lactam functionality. Abeta-lactam ring (β-lactam) is a cyclic amide comprising a heteroatomicring structure, consisting of three carbon atoms and one nitrogen atom.Non-limiting examples of beta-lactam antibiotics useful in aspects ofthe invention include penicillins, cephalosporins, cephamycins, penems,carbapenems, and monobactams. Beta-lactam antibiotics are effective (inthe absence of resistance) against a wide range of bacterial infections.The term “beta-lactam antibiotic” as used herein is considered toinclude any antibiotic undergoing mass or structural changes uponinactivation by an antibiotic resistant microorganism, provided saidmass or structural change can be detected by mass spectrometry.

The term “third generation cephalosporin” refers to such compoundsincluding, but not limited to cefixime, ceftazidime, cefotaxim,ceftriaxone, cefcapene, cefdaloxime, cefdinir, cefditoren, cefetamet,cefmenoxime, cefodizime, cefoperazone, cefotaxime, cefpimizole,cefpiramide, cefpodoxime, cefsulodin, cefteram, ceftibuten, ceftiolene,ceftizoxime, and oxacephem.

The term “beta-lactamase” means an enzyme (EC 3.5.2.6) produced by amicroorganism, preferably a bacterium, that has the ability to hydrolyzethe beta-lactam ring of beta-lactam antibiotics. Such enzymes are oftenclassified into 4 major classes (Classes A, B, C, and D) according tothe so-called Ambler classification scheme, based principally on proteinhomology. Examples of beta-lactamases include cephalosporinase,penicillinase, carbenicillinase, cloxacilanase, carbapenemase, andceftazidimase. It is meant that the term includes “normal”beta-lactamase, extended-spectrum beta lactamase (ESBL), as well as AmpCbeta-lactamase. Preferred beta-lactameses in aspects of the presentinvention are group A and D beta-lactamase enzymes according to theAmbler classification or are beta-lactam enzymes belonging to group 2according to the Bush classification (Bush et al. 1995. AntimicrobAgents Chemother. 39: 1211-33). Ambler class A antibiotics are theclassic active-site serine beta-lactamases and class D are a specificgroup of serine beta-lactamases that have little sequence similaritywith the class A beta-lactamases and are familiarly known as the OXA(oxacillinase) group. Also preferred is metallo-carbapenemase.

The term “extended-spectrum beta lactamase” (abbr. ESBL), as usedherein, initially called ‘extended-broad-spectrum beta-lactamase’, wasfirst coined for derivatives of TEM and SHV enzymes able to hydrolyseoxyimino-cephalosporins. These all belonged to beta-lactamase functionalgroup 2be. Subsequently, the term has been stretched to include: (i)enzymes with spectra similar to those of TEM and SHV mutants but derivedfrom other sources, e.g., the CTX-M and VEB types; (ii) TEM and SHVmutants with borderline ESBL activity, e.g., TEM-12; and (iii) variousbeta-lactamases conferring wider resistance than their parent types butnot meeting the definition for group 2be, e.g., OXA derivatives andmutant AmpC types with increased activity against cefepime.

The terms “resistant” and “resistance”, as used herein, refer to thephenomenon that a microorganism does not exhibit decreased viability orinhibited growth or reproduction when exposed to concentrations of theantimicrobial agent that can be attained with normal therapeutic dosageregimes in humans. It implies that an infection caused by thismicroorganism cannot be successfully treated with this antimicrobialagent.

The term “microorganism”, as used herein, refers in particular topathogenic microorganisms, such as bacteria, yeast, fungi and intra- orextra-cellular parasites. In preferred aspects of the present invention,the term refers to pathogenic or opportunistic bacteria. These includeboth Gram-positive and Gram-negative bacteria. By way of Gram-negativebacteria, mention may be made of bacteria of the following genera:Pseudomonas, Escherichia, Salmonella, Shigella, Enterobacter,Klebsiella, Serratia, Proteus, Campylobacter, Haemophilus, Morganella,Vibrio, Yersinia, Acinetobacter, Branhamella, Neisseria, Burkholderia,Citrobacter, Hafnia, Edwardsiella, Aeromonas, Moraxella, Pasteurella,Providencia, Actinobacillus, Alcaligenes, Bordetella, Cedecea, Erwinia,Pantoea, Ralstonia, Stenotrophomonas, Xanthomonas and Legionella. By wayof Gram-positive bacteria, mention may be made of bacteria of thefollowing genera: Enterococcus, Streptococcus, Staphylococcus, Bacillus,Listeria, Clostridium, Gardnerella, Kocuria, Lactococcus, Leuconostoc,Micrococcus, Mycobacteria and Corynebacteria. By way of yeasts andfungi, mention may be made of yeasts of the following genera: Candida,Cryptococcus, Saccharomyces and Trichosporon.

The term “mass spectrum”, as used herein, refers to a plot havingmolecular mass or a function thereof (e. g., mass-to-charge ratio (m/z),ion mass, etc.) as the independent variable. The dependent variable istypically a quantitative measure, such as abundance, relative abundance,intensity, concentration, number of ions, number of molecules, number ofatoms, counts/millivolt, counts, etc. For example, in the context ofions, a mass spectrum typically presents mass-to-charge ratio (m/z) asthe independent variable, where m is the mass of the ion species and zis the charge of the ion species, and the dependent variable is mostcommonly an abundance of each molecular ion and/or its fragment ions.The term “ion” means an atom or a group of atoms that has acquired a netelectric charge by gaining or losing one or more electrons or gaining orlosing one or more protons. An ion can be formed in numerous manners,including by breaking up a molecule of a gas under the action of anelectric current, of ultraviolet and certain other rays, and/or of hightemperatures.

The term “reference mass spectrum”, as used herein, refers to a controlmass spectrum intended for comperative analysis.

The term “substrate compound of a modifying enzyme”, as used herein,refers to any compound (antibiotic or not) that can be hydrolyzed by anantibiotic-modifying enzyme. The enzymatic modification of saidsubstrate will give rise to a reaction product with a differentmass-to-charge ratio (or mass spectrum) than the original substratecompound. The reaction product, in the case that the enzymaticconversion is a degradation, may be referred to herein as the“degradation product”.

The term “modifying enzyme”, as used herein refers broadly to anantimicrobial compound-modifying enzyme, such as for instance abeta-lactamase.

“Modification” as used herein refers to a chemical or physical(preferably chemical) alteration of the antimicrobial compound whichrenders the compound inactive with respect to its antimicrobialactivity. Modification may include degradation, which refers to thedeletion of chemical moieties from the compound molecule resulting in alower molecular mass, optionally in combination with an alteredmass-to-charge ratio. Alternatively, modification may includesubstitution or addition of chemical moieties on the compound molecule,thereby inactivating the compound with respect to its antimicrobialactivity, which mode of modification provides the molecule with analtered mass, optionally in combination with an altered mass-to-chargeratio.

The term “cell lysate” as used herein refers to cell suspensions orfractions thereof, obtained by disruption or lysing of the cells. Thecrude cell lysate contains all proteins, glycoproteins, polysaccharides,lipids, and nucleic acids. The cell lysate in aspects of the presentinvention may comprise whole cells, but will essentially consist ofparts of cells or any fraction or mixtures thereof obtained after alysis step. Cell lysate solutions, however, can include, withoutlimitation, a solution of lysed cells that is treated such that selectedmolecules are removed or rendered inactive. It follows that thissolution remains substantially “crude” with respect to most purifiedcellular constituents. For example, a cell lysate can be a solution oflysed cells that is treated with an agent that inactivates or removespolymerase inhibitors. In addition, a cell lysate can be a solution oflysed cells that is treated with an anti-coagulant. Any method can beused to lyse cells in a cellular sample. For example, osmotic shock,sonication, heating, physical disruption, microwave treatment, andenzymatic and/or alkaline lysis are methods that can be used to lysecells.

The term “growth medium”, as used herein, refers to a medium comprisingall the elements necessary for the expression of a metabolism and/or forthe growth of microorganisms. The growth medium may be solid, semi-solidor liquid. The growth medium may comprise one or more elements incombination, such as amino acids, peptones, carbohydrates, nucleotides,minerals, vitamins, active molecules such as antibiotics, enzymes,surfactants, buffers, phosphate salts, ammonium salts, sodium salts,metal salts, one or more substrates enabling the detection of an enzymeactivity, etc.

The term “supernatant”, as used herein, refers to the liquid suspensionremaining when cells grown in a liquid medium (e.g., a liquid broth) areremoved by centrifugation, filtration, sedimentation, or other meanswell known in the art, and containing dissolved and suspended material.

The terms “matrix material” and “MALDI matrix material”, as used herein,are interchangeable and refer to a compound, whether in solution orsolid, which may be used to form a matrix for use in MALDI massspectrometry. For MALDI, the analyte must be embedded in a large excessof molecules which are well-absorbing at the wavelength at which thelaser emits. These matrix molecules are generally small, organiccompounds, mainly acids. Appropriate matrix materials for each type oflaser used in MALDI are well known in the art and the term “MALDI matrixmaterial” will be clearly understood by one of skill in the art. Withoutlimiting the present invention, examples of commonly used matrixmaterials include sinapinic acid (SA), α-cyano-4-hydroxycinnamic acid(HCCA), 2,5-dihydroxybenzoic acid (DHB),7-hydroxy-4-(trifluoromethyl)coumarin (HFMC), 3-Hydroxy Picolinic Acid(3-HPA), 5-(trifluoromethyl)uracil, caffeic acid, succinic acid,anthranilic acid, 3-aminopyrazine-2-carboxylic acid,tetrakis(pentafluorfenyl)porfyrine and ferulic acid. Matrices aresuitably dissolved in acetonitrile/water/formic acid (500:500:1; v/v/v),or other suitable ratio's depending on the matrix used.

The term “sample”, as used herein, refers to a substance that containsor is suspected of containing an analyte, such as a microorganism orbeta lactamase to be characterized, or a beta-lactamase substrate or itsbeta-lactamase degradation product. A sample useful in a method of theinvention can be a liquid or solid, can be dissolved or suspended in aliquid, can be in an emulsion or gel, and can be bound to or absorbedonto a material. A sample can be a biological sample, environmentalsample, experimental sample, diagnostic sample, or any other type ofsample that contains or is suspected to contain the analyte of interest.As such, a sample can be, or can contain, an organism, organ, tissue,cell, bodily fluid, biopsy sample, or fraction thereof. A sample usefulin a method of the invention can be any material that is suspected tocontain analytes, such as substrates of beta-lactamases and ESBLs. In abiological context, a sample can include biological fluids, wholeorganisms, organs, tissues, cells, microorganisms, culture supernatants,subcellular organelles, protein complexes, individual proteins,recombinant proteins, fusion proteins, viruses, viral particles,peptides and amino acids.

The term “sample support”, as used herein, refers to all supports thatare suitable to receive a sample for MALDI MS analysis. Commonly usedare 10×10 stainless steel target plates (Perseptive Biosystems,Framingham, Mass., USA), if appropriate the target plates can behydrophobically coated.

The term “Michaelis-Menten constant”, often referred to as “Km”, as usedherein, refers to the substrate concentration at which the rate ofenzymatic reaction is half its maximum. The term “maximum reactionrate”, often referred to as “Vmax”, as used herein, refers to themaximum rate of an enzymatic reaction at saturating substrateconcentrations. Michaelis-Menten kinetics describe the rate ofproduction of molecules produced by enzymatic chemical reactions. Todetermine the maximum rate of an enzymatic reaction, the substrateconcentration is increased until a constant rate of product formation isachieved. This is the ‘maximum velocity’ (Vmax) of the enzyme. In thisstate, enzyme active sites are saturated with substrate. Since thesubstrate concentration at Vmax cannot be measured exactly, enzymes canbe characterized by the substrate concentration at which the rate ofreaction is half its maximum. This substrate concentration is referredto as the Michaelis-Menten constant (KM). For enzyme reactionsexhibiting simple Michaelis-Menten kinetics, this represents thedissociation constant (affinity for substrate) of the enzyme-substrate(ES) complex. Low values indicate high affinity.

The term “MALDI triple-quadrupole MS”, as used herein, refers to atechnique of matrix-assisted laser desorption/ionization wherein themass spectrometer has three quadrupoles arranged parallel to incomingions. The first quadrupole acts as a mass filter. The second quadrupoleacts as a collision cell where selected ions are broken into fragments.The resulting fragments are scanned by the third quadrupole. Quadrupolemass analyzers use oscillating electrical fields to selectivelystabilize or destabilize the paths of ions passing through a radiofrequency (RF) quadrupole field. Only a single mass-to-charge ratio ispassed through the system at any time, but changes to the potentials onmagnetic lenses allows a wide range of m/z values to be swept rapidly,either continuously or in a succession of discrete hops. A quadrupolemass analyzer acts as a mass-selective filter.

The term “quantifying”, as used herein, refers to any method forobtaining a quantitative measure. For example, quantifying amicroorganism can include determining its abundance, relative abundance,intensity, concentration, and/or count, etc.

The term “structural biomolecule”, as used herein, refers to any cellprotein, glycoprotein, polysaccharide, lipid, nucleic acid etc theamount of which is essentially constant between individual cells of aculture of microorganisms, and which can be used to quantify thosemicroorganisms. If DNA is used for instance, quantification can proceedvia DNA amplification or the use of (MS identifiable) nucleic acidprobes. Such methods for quantification may for instance use standardcalibration curves wherein DNA content is plotted against cell number oranother biomass parameter (such as optical density in culture or totalcarbon mass).

The term “metabolite”, as used herein, refers to a compound generated asa result of the functioning of a biochemical reaction in a cell ororganism the amount of which is essentially constant between individualcells of a culture of microorganisms, and which can be used to quantifythose microorganisms.

Preferred Embodiments

The invention provides in a method for characterizing the antibioticresistance of a microorganism. A first step in such a method is theprovision of one or more reference mass spectra of antibiotic compounds,suitable mimetic substrates thereof, or of molecular targets of theantibiotic compound for which resistance is to be characterized.Reference spectra can be produced by using any mass spectrometric (MS)technique that is to be used in the analysis of the samples. A preferredMS technique is MALDI-MS.

A suitable beta-lactamase substrate is any beta lactam antibiotic.Alternatively, beta-lactam derivatives or mimetics may be used thatinduce expression of the beta-lactamase in the microorganism, and/orthat are hydrolysed by enzymatic activity of the beta-lactamase. Themimetic substrates used in aspects of the invention themselves may, butneed not necessarily, exhibit any antibiotic activity.

Preferably, the beta-lactamase substrate is a compound of which thebeta-lactamase degradation product is readily discernable by MS,preferably such that the substrate and its degradation product havedifferent mass-to-charge ratios.

A further step in a preferred method for characterizing the beta-lactamantibiotic resistance of a microorganism involves the exposure of amicroorganism, a cell lysate thereof, or a growth medium supernatantthereof, to the substrate compound in aqueous liquid to thereby providean exposed sample.

A suitable exposed sample may be a body fluid or body tissue sample of asubject, i.e. a human or animal subject, suspected of carrying amicroorganism the beta-lactam antibiotic resistance of which is to becharacterized. Suitable body fluid samples may be blood, stool or urinesample.

The exposure of the microorganism to the substrate compound may thusinvolve in vivo or in vitro exposure.

Exposure may in certain embodiments comprise an incubation step whereinthe microorganism is incubated for a short period of time, for instancebetween 1-5 minutes and 1-3 hours in a solution containing theantimicrobial agents of interest. In addition, lysates of microorganismsand supernatants of microbial cultures can be used. When the specificenzymes are present, the antimicrobial agents or their mimeticsubstrates are modified or inactivated resulting in a differentcomposition in elements compared to the active drug form. This leads toa change in the mass of the antimicrobial agent that can be detected bymass spectrometry.

It is an advantage of the present invention that crude cell lysates canalso be used to provide an exposed sample. Hence, the microorganism thatis to be characterized needs no longer be viable, nor does the exposedsample need to be purified before any beta-lactamase activity thereincan be detected.

When a microorganism contains a beta-lactamase gene but does not producethe enzyme itself under the prevailing growth conditions, beta-lactamaseproduction in that organism may be induced by culturing themicroorganism in presence of a beta-lactam antibiotic or abeta-lactamase-inducing compound. Preferably, an optional step ofinducing or activating beta-lactamase production takes place prior tobacterial cell lysis.

In general, the capacity of the exposed sample to modify an antibioticcompound may be detected by detecting either a decrease in theantibiotic substrate compound (or its mimetic), or an increase in thereaction product of the hydrolysis reaction between modifying enzyme andsubstrate compound. Hence, the beta-lactamase activity in the exposedsample may be detected by detecting either a decrease in thebeta-lactamase substrate compound or an increase in the reaction productof the hydrolysis reaction between beta-lactamase and substratecompound.

Alternatively, the capacity of the exposed sample to modify anantibiotic compound may be detected by detecting a modification in themolecular target of the antibiotic compound. For instance, resistance toerythromycin, ciprofloxacin, vancomycin, methiciline and tetracycline isbased on target modification, such as RNA methylation. Also these targetmodifications may be detected by mass spectrometry as described herein.Hence, the present invention is not limited to the detection ofbeta-lactamases as modifying enzymes, and hence the characterization ofresistance to beta-lactams. Also other resistance to antibioticcompounds that is not based on drug modification can be characterizedusing the aspects of the present invention. Although beta-lacams usuallyinactivate the antibiotic drugs by hydrolysis, other types of enzymaticmodification can also be detected and characterized using the means andmethods of the present invention. For instance, aminoglycosides aremodified by the addition of a phosphate moiety. Such modifications ofthe substrate of the modifying enzyme can also be detected by themethods of the present invention.

It is an important finding of the present inventors that the change in(quantitative amount of) reaction or target compounds can be measuredvery accurately by mass spectroscopy. Hence, after the incubation step,the exposed sample is prepared for mass spectrometry using generic massspectrometry sample preparation protocols such as protein precipitationwith organic solvents, solid-phase extraction (SPE), or liquid-liquidextraction (LLE). Approximately 1 μL of the prepared solution is usedfor the mass spectrometric analysis. In preferred embodiments of thepresent invention, MALDI MS is used and more preferably MALDI quadrupoleMS is used. Using MALDI MS, the reaction compounds can be measured withsuch accuracy that exposure times (incubation periods) can be veryshort. Successful characterizations have been attained with anincubation time of about 5 minutes.

Maldi MS involves applying the exposed sample together with a matrixmaterial to a mass spectrometric sample support and drying the sample onthe sample support to produce a mass spectrometric sample. Suitablematrix materials are indicated herein above, and the nature of thematrix material is not particularly limiting. The preparation of themass spectrometric sample from the exposed sample can be performed bymethods known per se to one of skill in the art of mass spectrometry.

Once the sample is mounted in the mass spectrometer, the mass spectrumof the sample is acquired by standard procedures that depend on the typeof equipment and MS methods used.

In a method of the invention, the step of detecting substrate or targetmodification (such as beta lactamase substrate degradation or RNAmethylation) is performed by using MS, preferably by tandem massspectrometry (MS-MS) or by matrix-assisted laser desorption/ionization(MALDI). Mass spectrometry provides a powerful means of determining thestructure and identity of complex organic molecules, including proteinsand peptides. In MS, a sample compound is bombarded with high-energyelectrons causing it to fragment in a characteristic manner. Thefragments, which are of varying weight and charge, are then passedthrough a magnetic field and separated according to their mass-to-chargeratios. The resulting characteristic fragmentation signature pattern ofthe sample compound (the mass spectrum) is used to identify andquantitate that compound. A typical MS procedure comprises the followingsteps:

-   -   1. loading of a sample onto the MS instrument, by applying the        sample optionally (in case of a special form of MS called MALDI)        together with a matrix on a mass spectrometric sample support        and drying the sample or the mixture on the support by        evaporation of the solvents.    -   2. ionizing the components of the sample by one of a variety of        methods (e.g., by impacting them with an electron beam), which        results in the formation of charged particles (ions)    -   3. accelerating the positive ions by an electric field    -   4. computating the mass-to-charge ratio (m/z) of the particles        based on the details of motion of the ions as they transit        through electromagnetic fields, and    -   5. detecting the ions, which in step 4 were sorted according to        m/z.

In MALDI MS, the matrix consists of crystallized molecules, of which thethree suitable examples are 3,5-dimethoxy-4-hydroxycinnamic acid(sinapinic acid), α-cyano-4-hydroxycinnamic acid (alpha-cyano oralpha-matrix) and 2,5-dihydroxybenzoic acid (DHB). The matrix solutionis mixed with the exposed sample. The organic solvent allows hydrophobicmolecules to dissolve into the solution, while the water allows forwater-soluble (hydrophilic) molecules to do the same. This solution isspotted onto a MALDI plate or support (usually a metal plate designedfor the purpose). The solvents evaporate, leaving only therecrystallized matrix, together with the sample molecules dispersedthroughout the matrix crystals.

Suitable MS applications that can be used in aspects of the inventioninclude MALDI-TOF MS mass spectrometry, MALDI-FT mass spectrometry,MALDI-FT-ICR mass spectrometry, MALDI Triple-quadrupole massspectrometry. Using MALDI-TOF mass spectrometry, the throughput isestimated to be 1 minute per sample. Using MALDI-triple quadrupole massspectrometry, the duration of the test can be decreased to approximately5 seconds per sample without loss of sensitivity or specificity.

Following the acquisition of the mass spectra, the sample-derived massspectra are compared with the reference mass spectra of theantimicrobial compound, its enzymatic modification product, itsmolecular target, or of a substrate compound of a its modifying enzymein a qualitative, semi-quantitative or quantitative manner By suchcomparison, the qualitative, semi-quantitative or quantitative presenceof modification of the substrate or the target and/or the production ofmodification products can be determined.

Both the inactivated or modified antibiotic (e.g. degradation product)and intact antibiotic substrate (or the mimetic substrate), as well asthe molecular target can be measured simultaneously by massspectrometry, and the ratio of product-to-substrate can for instance betaken as a measure of the microorganism's ability to inactivate ormodify the tested substrates. Alternatively or in addition, a decreasein the level of the substrate alone or an increase in the level of theproduct alone in the sample can be used as a measure of theantibiotic-inactivating or antibiotic-modifying ability of themicroorganism. Alternatively, an increase in the level of the moleculartarget, or an increase in the level of the modified (resistant) target,may be taken as an indication of the microorganism's resistance. In caseof characterization of antibiotic resistance against drugs involvingtarget modification as resistance mechanism, the step of exposing amicroorganism, a cell lysate thereof, or a growth medium supernatantthereof, to an antimicrobial compound in aqueous liquid will amount tothe provision of a sample of the microorganism, a cell lysate thereof,or a growth medium supernatant thereof, and the detection therein ofmodified targets.

Hence, in one embodiment of aspects of the invention, modification ofthe antimicrobial substrate compound is taken as proof for theproduction of for instance a beta lactamase by the microorganism, andindicates that the microorganism is likely to be resistant to thebeta-lactam antibiotic compounds that are inactivated, for instance bydegradation, by that specific beta lactamase for which an antimicrobialsubstrate compound or suitable mimetic substrate was provided. In thisway, the resistance of said microorganism to for instance a beta-lactamantibiotic may be characterized.

Alternatively, in another embodiment of aspects of the invention, thepresence of a modification (in relative amount or in chemicalcomposition) of the molecular target of the antimicrobial compound inthe microbial cell is taken as an indication that the microorganism islikely to be resistant to the antibiotic compound of interest. In thisway, the resistance of said microorganism to for instance erythromycin,ciprofloxacin, vancomycin, methiciline and tetracycline may becharacterized.

In aspects described above, the invention provides in certainembodiments a method for the rapid diagnosis of microorganisms thatproduce enzymes that inactivate or structurally modify antimicrobialagents. The method can be used for rapid detection of ESBL activity.Especially in a hospital setting, this is highly needed because thirdgeneration cephalosporins are widely used in the empiric therapy forseriously ill patients with infections. Rapid detection of ESBL activityin a patient's sample is of importance to start as early as possiblewith the most appropriate antibiotic drug therapy for that patient. Themethods of the present invention can be used for rapid detection of ESBLactivity. Furthermore, the methods of the present invention can beapplied on supernatants of microbial cultures or on microorganismsisolated directly from a patient's sample, e.g. after centrifugation ofurine samples. In this way, it should be possible to detect ESBLactivity even sooner than detection of the (cultured) bacteriathemselves.

Mass spectrometry has not been used to detect enzymatic inactivation orchemical modification of antibiotics by monitoring the decrease insubstrate intensity and/or the increase in product intensity.Furthermore, mass spectrometry has not been used to study enzymeactivity in complex samples such as lysed microorganisms. More inparticular, the specific detection and characterization of ESBL enzymesby MS has never been reported before.

A diagnostic method for rapid detection of ESBL activity suitablycomprises the liberation from the microorganisms of the beta lactamaseenzymes that inhibit the antimicrobials by lysing the sample using alysis reagent. Subsequently, these lysates may be transferred,preferably using an automated pipetor, into a multiwell strip, such asthe ATB™ or Rapidec™ strips, which strips contain wells with reagentsfor performing different test reactions based on different antimicrobialcompounds. Some of the wells contain a certain amount of one or severalbeta lactamase substrates, for instance in a dried or immobilized(glued) form. Some of the wells could also contains one or severalinternal standard(s) for easing quantification. Some wells could also beused as control without substrates for self-degradation of theantimicrobial compounds.

Following the transfer into the wells, and the incubation therein for ashort period of time as indicated herein, the exposed sample of eachwell may suitably be put on a MALDI plate or any other MS support. Inthe case of MALDI, a suitable matrix material is added to the support.Thereafter, the mass spectra are acquired. Spectral analysis is suitablyperformed using dedicated analysis algorithms. The spectra as obtainedfrom the exposed sample are then compared with reference spectra usingcomputer software in order to determine the presence of degradation ofthe substrate for each well. In case of degradation, dedicated softwaremay provide the test results in a report, which report may include, forinstance: (1) the identity (species name) of the microorganism (whichidentification may be brought about by reference tests, optionallyavailable in the same or a parallel teststrip), (2) a list of testedantimicrobials as provided in the multiwall test strip, (3) a list ofantimicrobials inhibited or degraded by the microorganism, (4) theresistance mechanism(s) supposed to be responsible of antimicrobialinhibition, (5) dedicated interpretation comments with regards to theresults.

Alternatively, the step of exposing a microorganism, a cell lysatethereof, or a growth medium supernatant thereof, to said substratecompound in aqueous liquid (to thereby provide an exposed sample), maybe performed on the mass spectrometric sample support. Upon allowing theoptionally present beta lactam enzymes to degrade the substrate compoundof a beta-lactamase enzyme on the sample support the MALDI matrix may beadded to the exposed sample directly.

A method of the present invention can be performed using complexsamples, including crude cell lysates or patient samples. The methodallows for the precise assessment of molecules in the size range ofantimicrobial agents (normally between 200 and 1000 daltons) and cansuitably be used to determine the activity of antibiotic-inactivating orantibiotic-modifying enzyme, for instance using antimicrobial drugs assubstrates.

A method of the present invention can also be used as ESBL confirmatorytest. In most clinical microbiology laboratories bacteria are firstscreened for the ESBL phenotype and then the ESBL phenotype is confirmedusing a separate ESBL phenotypic confirmatory test. Aspects of thepresent invention can be used to confirm the ESBL phenotype in bacteria.The potential advantage of a method of the present invention overcurrent phenotypic tests is that the method proposed herein not onlyworks with bacterial suspensions but also with lysates of bacteria. Theuse of bacterial lysates neutralizes the potential bias due toresistance based on other mechanisms, in particular decreased influx andincreased efflux of drugs. Use of bacterial lysates is incompatible withcurrent phenotypic ESBL confirmatory tests, because these tests rely onbacterial growth.

A method of the present invention can also be used as high throughputscreening method for new beta-lactamase inhibitors in the pharmaceuticalindustry. Currently, the beta-lactamase inhibitors clavulanic acid andtazobactam are combined with amoxicillin and piperacillin respectivelyto overcome the problem of beta-lactamase producing bacteria. Theclavulanic acid or tazobactam inhibit the activity of thebeta-lactamases while the amoxicillin or piperacillin kill the bacteria.Considering the growing problem of ESBL, the pharmaceutical industryneeds tools to screen for novel compounds that inhibit ESBLs. Thepresent invention is highly suited for this purpose.

EXAMPLES

We have detected beta-lactamase activity in crude lysates of E. coliproducing CTX-M-1 and CTX-M-9 and in K. pneumoniae producing SHV-2 usingbenzylpenicillin as substrate. We were able to monitor the enzymekinetics of pure penicillinase (from B. cereus) using benzylpenicillinas substrate.

Although the present invention has been illustrated and described withrespect to several preferred embodiments thereof, various changes,omissions and additions to the form and detail thereof, may be madetherein, without departing from the spirit and scope of the invention.

What is claimed is:
 1. A method for characterizing the antibioticresistance of a microorganism, said method comprising the steps of: a)providing a reference mass spectrum of an antimicrobial compound, itsenzymatic modification product, its molecular target, or of a substratecompound of a its modifying enzyme; b) exposing a microorganism, a celllysate thereof, or a growth medium supernatant thereof, to saidantimicrobial compound or said substrate compound in aqueous liquid tothereby provide an exposed sample; c) acquiring a mass spectrum of theexposed sample; d) comparing the mass spectrum acquired in step c) withthe reference mass spectrum of step a), and e) determining from saidcomparison whether modification of said antimicrobial compound, itsmodification product or its molecular target or of said substrate hasoccurred following said exposure, and establishing that saidmicroorganism is potentially resistant to said antimicrobial compoundwhen said modification is observed.
 2. Method according to claim 1,wherein said modification comprises enzymatic inactivation or enzymaticdegradation of said antimicrobial compound or said substrate, and/ormethylation or overproduction of its molecular target.
 3. Methodaccording to claim 2, wherein said enzymatic degradation is due todegradation by a beta-lactamase.
 4. Method according to claim 3, whereinsaid beta-lactamase enzyme is selected from group A and D beta-lactamaseenzymes according to the Ambler classification and beta-lactamaseenzymes belonging to group 2 according to the Bush classification. 5.Method according to claim 4, wherein said beta lactamase enzyme is anextended-spectrum beta lactamase (ESBL).
 6. Method according to any oneof the preceding claims, wherein said microorganism is a suspectedESBL-producing microorganism, preferably selected from Klebsiellapneumoniae, Escherichia coli, Klebsiella oxytoca and Proteus mirabilis.7. Method according to claim 1, wherein said antimicrobial compound is abeta-lactam antibiotic, preferably selected from the group consisting ofpenicillins, cephalosporins, cephamycins, and carbapenems, morepreferable selected from the group consisting of ceftazidime,cefotaxime, ceftriaxone, cefpodoxime, and aztreonam.
 8. Method accordingto claim 1, wherein said method is part of a method for characterizationof an antibiotic modifying enzyme of a microorganism, preferably saidantibiotic modifying enzyme is an extended-spectrum beta lactamase(ESBL) enzyme.
 9. Method according to claim 8, wherein said method forcharacterization of said enzyme comprises the determination of the rateof modification, preferably degradation, of said antimicrobial compoundor said substrate compound and/or the rate of production of theenzymatic modification product of said compound or substrate or of themolecular target of said compound to thereby determine theMichaelis-Menten (Km) constant and maximum reaction rate (Vmax) for saidenzyme.
 10. Method according to claim 1, wherein after step (b) saidexposed sample is applied together with a matrix material to a massspectrometric sample support and wherein said sample is dried on thesample support to produce a mass spectrometric sample formatrix-assisted laser desorption ionization mass spectrometry(MALDI-MS).
 11. Method according to claim 10, wherein said mass spectraare acquired using MALDI triple-quadrupole MS.
 12. Method according toclaim 1, wherein said exposed sample is an antimicrobialcompound-exposed crude cell lysate of said microorganism.
 13. Methodaccording to claim 1, wherein in step (b) said microorganism isquantified by quantifying in said samples one or more structuralbiomolecules or metabolites derived from said microorganism, preferablywherein said structural biomolecules or metabolites are DNA molecules.14. Kit of parts for characterizing the antibiotic resistance of amicroorganism comprising: a) a lysis buffer for lysing a microorganism;b) at least one antimicrobial compound or a substrate of anantimicrobial compound-modifying enzyme, and c) a MALDI matrix material,preferably said kit of parts further comprising: d) a carrier carryingsaid at least one antimicrobial compound or substrate, wherein saidcarrier is optionally in the form of a disposable mass spectrometricsample support.
 15. A system for characterizing the antibioticresistance of a microorganism comprising: at least one antimicrobialcompound or a substrate of an antimicrobial compound-modifying enzyme; acontainer for exposing a microorganism, a cell lysate thereof, or agrowth medium supernatant thereof, to said at least one antimicrobialcompound in aqueous liquid, preferably wherein said at least onesubstrate compound is provided in said container; a lysis buffer forlysing said microorganism; a MALDI matrix material; a mass spectrometrydevice; a reference mass spectrum of an antimicrobial compound, itsenzymatic modification product, its molecular target, or of a substratecompound of a its modifying enzyme, and a mass spectrometric samplesupport, optionally further comprising an automated pipetor for liquidhandling; a computer program comprising computer program code adapted toperform the steps of a) providing a reference mass spectrum of anantimicrobial compound, its enzymatic modification product, itsmolecular target, or of a substrate compound of a its modifying enzyme;b) exposing a microorganism, a cell lysate thereof, or a growth mediumsupernatant thereof, to said antimicrobial compound or said substratecompound in aqueous liquid to thereby provide an exposed sample; c)acquiring a mass spectrum of the exposed sample; d) comparing the massspectrum acquired in step c) with the reference mass spectrum of stepa), and e) determining from said comparison whether modification of saidantimicrobial compound, its modification product or its molecular targetor of said substrate has occurred following said exposure, andestablishing that said microorganism is potentially resistant to saidantimicrobial compound when said modification is observed. when saidprogram is run on a computer or wherein said computer program isembodied on a computer readable medium.
 16. (canceled)
 17. (canceled)